This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Transcription plays a central role in gene expression and is a prime target for regulatory processes. Important among these is transcriptional pausing, which plays a regulatory role in both prokaryotes and eukaryotes. For example, by pausing RNAP at key sites, pausing allows RNAP to interact with or recruit regulators in response to environmental cues, or ensures proper folding of RNA transcripts. Pausing couples translation and transcription in bacteria;it also is a prerequisite for intrinsic as well as Rho-dependent termination of transcription. Three classes of pausing exist: similar to intrinsic termination, class I pauses require the formation of a stem-loop hairpin structure in the nascent RNA transcript that interacts with the RNAP;class II pauses are stabilized by backtracking of RNAP along the RNA and DNA;and class III pauses are stabilized by protein factors. Class I (hairpin-dependent) pausing can be prolonged by interaction of the elongation complex with NusA. NusA is also involved in enhancing termination efficiency at intrinsic terminators. Despite its central regulatory role, the molecular mechanisms of transcriptional pausing are unknown. A common intermediate state of RNAP has been suggested to lead to either transcriptional termination, class I or class II pausing, or transcriptional arrest. By studying the structure of an RNAP elongation complex trapped at a class I pause site and in the absence or presence of NusA, it will be possible to gain insights into this fundamental process. While pausing is a fundamental event in regulating transcript elongation, the underlying mechanism inducing the paused state is poorly understood. Crosslinking, biochemical, and mutagenesis experiments point to rearrangements of the RNAP active site induced by interactions with the pause hairpin more than 50 [unreadable] away in the RNA exit channel. High-resolution crystal structures of paused RNAP elongation complexes will elucidate these rearrangements and how they are induced by hairpin binding.